The goal of these studies is to determine the required biochemical events that occur between tumor promoter-receptor interaction and the activation of effectors of neoplastic transformation. Candidate second messengers include protein phosphorylation by protein kinase C (PKC) and epidermal growth factor (EGF) receptor kinase and kinase-regulated trans-activation of gene expression. A putative C-kinase substrate of 80-kDa has been found to be differentially phosphorylated in promotion-resistant (P-), -sensitive (P+), and neoplastically transformed JB6 mouse epidermal cells, with little or no phosphorylated 80-kDa phosphoprotein (pp8O) seen in transformed cells. Western analysis indicates that p8O is regulated at the level of synthesis with little or no p8O protein detectable in transformed JB6 cells. A CDNA clone of p8O has recently been isolated by screening a JB6 P- library with p80 antibody. This p80 cDNA, when used as a probe, detects a 2.8-Kb RNA that progressively decreases during the progression from P- to transformed (Tx) phenotype in certain cases but does not generalize to multiple independent cells of each phenotype. When compared with GenBank the sequence emerges as novel. Recent studies on 12-0-tetradecanoyl-phorbol-13-acetate (TPA)-inducible genes have focused on those regulated by the trans-acting transcriptional factor AP-1 (Jun/Fos Complex). The tumor promoters TPA and EGF induce AP-1-regulated gene expression in P+ but not P- JB6 cells. This suggests that AP-1-regulated gene expression may be required for tumor promoter-induced transformation. The mechanism of differential trans-activation and transformation by TPA appears to involve differential basal and induced levels of c-jun mRNA and Jun protein but does not involve differential induction of c-fos, fra, jun D, or jun B. A c-jun differential was not found after EGF treatment thus suggesting that TPA and EGF induce AP-1-dependent trans-activation of gene expression by different pathways. Finally, the phosphorylation of c-jun and fra-1 is differentially regulated in P- and P+ cells, suggesting a second promotion-relevant mode of regulation.